1. Field of the Invention
This invention relates to an improved radioimmunoassay for detecting cannabinoids. More particularly, this invention is directed to novel .sup.125 I-labeled THC derivatives, novel compounds yielding antibody, the use of said THC derivatives in the radioimmunoassay for THC and the use of an anion exchange resin for the separation of bound from unbound antigen.
2. Description of the Prior Art
For the past several years, there has been an increasing demand for qualitative and quantitative assays for identifying and measuring the constituents of marijuana in the human body. The demand for these tests is due to the need to better understand the pharmacology of this drug and to determine the presence of cannabinoids in the biological fluids of suspected abusers.
Initial attempts to determine the presence of marijuana lacked sufficient sensitivity because the primary, active constituent, .DELTA.-9-tetrahydrocannabinol, is distributed and metabolized. Experiments with humans showed that, following almost complete metabolism, the metabolites were excreted, mainly via feces. Only small quantities of the hydroxy metabolite were noted in the urine.
The analytical methods presently available for the determination of cannabinoids in biological fluids include chromatographic, spectrometric and immunologic techniques. All of the methods are of value, especially when one method is used to cross-check the results of another technique. However, each of these types of techniques have certain drawbacks.
The gas and the liquid chromatographic methods require pre-treatment and/or purification to remove the cannabinoids from endogenous substances in the specimen which might interfere in the assay. The mass spectroscopic technique, in conjunction with gas chromatography (GC-MS), also requires sample pre-treatment. In addition, because of the size, cost and complexity of GC-MS, it is not ideal for application to routine screening.
With respect to the radioimmunoassay, the highly-lipophilic nature of the cannabinoid molecule can cause difficulties in the system in which the radioimmunoassay is performed. The cannabinoid may adhere to the glass or plastic container or the available protein in preference to remaining dissolved in the aqueous medium. Consequently, the separation of the bound from the free radiosotopically-labeled tetrahydrocannabinol derivative, which separation is the basis for the subsequent dose response measurement, can only be accomplished by judicious selection of test conditions.
To date, the radioimmunoassays for tetrahydrocannabinol have primarily employed tritiated tetrahydrocannabinol moieties for radioactive detection and charcoal or dextran-coated charcoal for separation of the bound from free antigen. For example, J. D. Teale et al., J. Pharm. Pharmac. 27, p. 465 (1975), S. J. Gross et al., Nature 252, p. 581 (1974) and Clarence E. Cook et al., NID Research Monograph 7, p. 15 (1976) all disclose the use of tritiated tetrahydrocannabinol moieties and charcoal and/or dextran-coated charcoal for the separation of bound from free antigen. In a paper presented at the 173rd National ACS Meeting, New Orleans, March 20-25, 1977, C. E. Cook presented a paper entitled "Radioimmunoassays of Cannabinoid Compounds" in which the use of tritiated and .sup.125 I-labeled tetrahydrocannabinol derivatives were used to develop radioimmunoassay procedures. Upon further investigation of the contents of this paper, it was found that the .sup.125 I-labeled tetrahydrocannabinol derivatives utilized had the following chemical structure: ##STR1##
The foregoing moiety was derived from the reaction of a "cold" (i.e., nonradioactive) iodinated compound with radioactive NaI* to effect a substitution of the radioactive iodine for the "cold" iodine according to the following reaction: ##STR2##
Once again, charcoal was used to separate the bound from free antigen.
In accordance with the present invention, a radioimmunoassay for tetrahydrocannabinol is disclosed wherein .sup.125 I-labeled tetrahydrocannabinol and novel .sup.125 I-labeled tetrahydrocannabinol antigens are mixed with fixed amounts of the specimen to be assayed followed by separation of bound from unbound antigen by use of an anion exchange resin.
The direct reaction of the foregoing radioactive antigens with antibody, followed by separation of bound from unbound antigen with an anion exchange resin, allows for a considerable reduction in the time necessary to run the assay without sacrificing the sensitivity of the test. For example, when operating according to the method of the claimed invention, the entire assay can be run in less than one hour as compared with the five hours which have been necessary in the prior art. Additionally, whereas prior art disclosures have required careful control of temperature at about 5.degree. C., there is no such requirement in the practice of the present invention.